Deoxyribonucleic acid, or DNA, is the genetic material in the nuclei of all cells. DNA is made of chemical building blocks called nucleotides. Nucleotides comprise three parts: a phosphate group, a sugar group and one of four types of nitrogen bases. The four types of nitrogen bases found in nucleotides are: adenine (A), thymine (T), cytosine (C) and guanine (G). To form a strand of DNA, nucleotides are linked into chains, with the phosphate and sugar groups alternating.
The structure of a DNA molecule was first described in 1953 by Francis Crick and James D. Watson as two strands wound around each other in a double helix to resemble a twisted ladder. The two strands of DNA contain complementary information: A forms hydrogen bonds only with T, C only with G. The precise order in which the four types of nitrogen bases (A, T, C, G) appear in the strand determines genetic characteristics of all life forms. The process of determining the precise order of nucleotides within a DNA molecule is termed DNA sequencing. DNA sequencing may be used to determine the sequence of individual genes, larger genetic regions (i.e. clusters of genes or operons), full chromosomes or entire genomes. Depending on the methods used, sequencing may provide the order of nucleotides in DNA or RNA isolated from cells of animals, plants, bacteria, archaea, or virtually any other source of genetic information. The resulting sequences may be used by researchers in molecular biology or genetics to further scientific progress or may be used by medical personnel to make treatment decisions.
Conventional methods of DNA sequencing comprise chemical (i.e. electrophoresis), optical, and electronic methods. As compared to other methods for detecting a DNA sequence, electronic sequencing methods differ from other sequencing technologies in that modified/labeled nucleotides and/or optics/optical measurements are not necessary. Instead, electronic sequencing methods rely on ion or other reaction byproducts to identify the relevant DNA sequence.
A type of electronic DNA sequencing, ion semiconductor sequencing is a method of DNA sequencing based on the detection of ions (for example, hydrogen ions) that are released during the polymerization of DNA. Ion semiconductor sequencing is a method of “sequencing by synthesis,” during which a complementary strand is built by incorporation (pairing of bases) based on the sequence of a template strand.
The incorporation of a deoxyribonucleotide triphosphate (dNTP) into a growing DNA strand involves the formation of a covalent bond and the release of pyrophosphate and a positively charged hydrogen ion. A dNTP will only be incorporated if it is complementary to the leading unpaired template nucleotide. Ion semiconductor sequencing leverages this process by detecting whether a hydrogen ion is released when a single species of dNTP is provided to the reaction.
Hydrogen ions may be detected by providing an array of microwells on a semiconductor chip. Beneath the layer of microwells is an ion sensitive layer, below which is an ion sensitive (ISFET) or a chemical sensitive (chemFET) sensor. Each microwell on the chip may contain a template DNA molecule to be sequenced. Each microwell containing a template strand DNA molecule also contains a DNA polymerase. A DNA polymerase is a cellular or viral enzyme that synthesizes DNA molecules from their nucleotide building blocks. A microfluidics device may be used to introduce a solution of unmodified A, T, C, or G dNTP into the microwells one after the other and one at a time. If an introduced dNTP is complementary to the next unpaired nucleotide on the template strand, a biochemical reaction occurs (which includes the release of a hydrogen ion) and the introduced dNTP is incorporated into the growing complementary strand by the DNA polymerase. If the introduced dNTP is not complementary there is no incorporation and no biochemical reaction. The release of a hydrogen ion during incorporation causes a change in the pH of the solution in the microwell. That change in the pH of the solution can be detected/measured by the ISFET or chemFET sensor and translated into an electrical pulse.
Before the next cycle of dNTP is introduced into the microwells, the microwells are flushed with a wash solution. Unattached dNTP molecules are washed out during the flush cycle. The detected series of electrical pulses are transmitted from the chip to a computer and are translated into a DNA sequence. Because nucleotide incorporation events are measured directly by electronics, intermediate signal conversion is not required. Signal processing and DNA assembly can then be carried out in software.
The chip may be fabricated by taking advantage of conventional semiconductor technology. However, the release of a hydrogen ion produces a small and transient signal that is difficult to measure. To further complicate detection and nucleic acid sequencing, ion detection is sensitive to various forms of contaminants on the chip surfaces. Accordingly, problems arise during manufacturing, packaging and exposure of the chip surfaces to the environment. Thus, there is a need for improved methods and an apparatus to prevent contamination of the chip surfaces for reliable ion detection and sequencing.